JPH05501204A - Diagnosis of glomerulonephritis - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Diagnosis of glomerulonephritis Background of the invention The present invention relates to the diagnosis of glomerulonephritis.

Glomerulonephritis is a kidney disease characterized by bilateral inflammatory changes within the glomeruli of the kidney. It is. Rapidly progressive glomerulonephritis (RPGN) can be caused by any of several underlying symptoms. immunodeposit or non-immune deposits are insufficient for the symptoms. Necrotizing and/or crescentic glomerulonephritis (immunodeficiency) with kimono -immune) NCGN), anti-globe basement membrane nephritis (anti-BM nephritis), I gA nephrobanosis, lupus nephritis, and post-hemolytic streptococcal glomerulonephritis. nothing. In addition, certain diseases other than glomerulonephritis, such as hemolytic uremic syndrome or acute Interstitial nephritis may result in a diagnostic picture of RPGN. Immune deficiency NCGN is renal Can be confined to the viscera (early NCGN) or sea urchin granulomas or microscopic nodules Vines associated with a systemic disease often classified as polyarteritis.

Diagnosis of conditions that cause RPGN may lead to recommendations for preventing or reversing worsening renal function. This is essential for initiating appropriate treatment. Kidney biopsy for RPGN patients It has been considered to be the most reliable treatment. However, this method is dangerous. This can be dangerous, and sometimes fail to provide reliable treatment, but this is because the focus of NCGN is often the cause. It is often localized and due to lack of small pieces of biological tissue specimens (Madio). dio), Kidney Int, 38:529.1990). Furthermore, the initial N Some symptoms of CGN cannot be clearly classified.

Serological tests have diagnostic value in some forms of RPGN. In particular, R.P. Patients with GN often have circulating autoantibodies against neutrophils and monocytes. these The presence of autoantibodies, usually referred to as anti-neutrophil cytoplasmic antibodies (ANCA), is a diagnostic tool. has been used as. ANCA was determined as a substrate by indirect immunofluorescence assay. Detected using ethanol-fixed normal human neutrophils.

Two staining patterns are described: (1) cytoplasmic and (2) nuclear or perinuclear. (Andrassy et al., Nephron 49.257- 258.1988). The cytoplasmic pattern is similar to that of active sea urchin granulomas. (Van der Woude et al.) , Lancet 1:806.1985), early NCGN or microscopic nodular polymorphism. Occasionally seen in other patients with onset arteritis (Jennett e) et al., Am, J., Pathol, 135:921.1987, Cohen (Co. hen) et al., Kidney Int, 37:799.1990). Cytoplasmic staining pa Turn-associated autoantibodies are found in the 27-29 key located in the secondary or secondary granule fraction. It is a soluble protein of rhodolton (kD) (Gross et al., Lan cet 1:1488.1987: Goldschm (eding), Kidney Int, 32 Near 79, 1987). in contrast, Nuclear or perinuclear staining patterns are present in only a few patients diagnosed with sea urchin granuloma. Found only in %. An antigen that is artificially redistributed in the production of neutrophils. often caused by antibodies against certain myeloperoxidases (MPOs) .

Ruko's pattern (Folk et al., N. Eng, J. Med, 31 8:1651) in some patients with early NCGN or microscopic polyarteritis nodosa. (Andrassy et al., CI in, Nep. hr.

1.32:159, 1989; Gans et al., Lancet 1:26 9.1989). Because of these obvious staining patterns, indirect immunofluorescence assays can be a useful diagnostic tool. However, analysis of staining patterns is difficult; and - at least 1000 sera before quantifying and interpreting human staining patterns. It is recommended to test the sample (Rasmussen et al., L. ancet 1 near 06.1988). can be quantified more simply and more than 5 times more easily. Assays for autoantibodies associated with these conditions provide a faster and more accurate diagnosis. and encourage early treatment involvement. Accurate diagnosis is especially important; Treatment for these diseases involves potentially harmful drugs, and doctors without a definitive diagnosis This is because patients may dislike treatment. A more accurate diagnosis is It may also provide data that contribute to understanding the pathogenesis of related diseases.

Regarding quality (p. 29). The protein is approximately 2 It has a size of 9 kD and can bind to isopropyl fluorophosphate, It has an isoelectric point of about 92 to about 9.4 and is present in the serum of patients with sea urchin granuloma. can bind to autoantibodies, and the N-terminal amino acid sequence 11e-Val-Gly -Gly-His-GIU-^1a-Gln-Pro-! Ti5-3er-^r g-Pro-Tyr-Met-^1. a-3er-Leu-Gin-Met-^ rg|Gly-Asn-Pr o-Gly-Set-His (SEQ ID NO: 1). Substantially pure means A protein that is 95% or more pure in quantity and in which the protein is bound in its natural state, - This means that it is substantially free of fat and carbohydrates.

In another aspect, the invention provides monochromes capable of forming immune complexes with p29. Regarding local antibodies.

In another aspect, the present invention provides a method for detecting autoantibodies useful in the diagnosis of sea urchin granuloma. Regarding how to This method involves the step of contacting the biological fluid to be tested with p29. Including floors. Any immune complexes formed are helpful in diagnosing Urchinona granuloma , detected and used as an indication of the presence of autoantibodies in biological fluids. It will be done.

In another aspect, the present invention provides a method for detecting autoantibodies useful in the diagnosis of sea urchin granuloma. Regarding how to This method = (a) the monoclonal antibody of the present invention and the monoclonal antibody; (b) providing an immune complex with an antigen that reacts with the local antibody; and (b) the biology being tested. and (C) assisting in the diagnosis of Wenner's granuloma. One example is the presence of autoantibodies in biological fluids. detecting the binding of autoantibodies to the autoantibodies.

In another aspect, the present invention provides a vector comprising a DNA sequence encoding p29 protein. - related.

In another aspect, the invention provides immunodeficiency necrosis and/or crescentic glomerulonephritis. Concerning a method for detecting autoantibodies useful for diagnosis. This method = (a) protein of the present invention (b) contacting the biological liquid to be tested with the substance and the biological liquid to be tested; (C) contacting the biological fluid to be tested; and (C) step (a) or step (b). The method consists of detecting immune complexes formed in the immune system. Autoantibody indicators useful in the diagnosis of epinephrine necrosis and/or crescentic glomerulonephritis becomes.

In another aspect, the invention provides immunodeficiency necrosis and/or crescentic glomerulonephritis. The present invention relates to a method for detecting autoantibodies useful for diagnosis. This method includes (a) the method of the present invention; Providing an immune complex of a monoclonal antibody and an antigen that reacts with the monoclonal antibody. and (b) react with myeloperoxidase. (C) providing an immunoconjugate with a monoclonal antibody; (d) contacting the biological fluid to be tested with the immune complex of step (b); (e) the immune complex of step (a) or (b); step (a) or (b) of detecting the binding of autoantibodies to Binding of autoantibodies to the epidermis complex may lead to immunodeficiency necrosis and/or crescentic threads. It is an indicator of the presence of autoantibodies, which is useful in the diagnosis of bulbar nephritis.

In another aspect, the invention provides diagnostic methods for the detection of autoantibodies in biological fluid samples. the autoantibodies are associated with immunodeficiency necrosis and/or crescent-shaped globules. The kit is useful for diagnosing systemic nephritis, and the kit contains p29 protein and miniloba-oxidase. including.

In another aspect, the invention provides diagnostic methods for the detection of autoantibodies in biological fluid samples. the autoantibodies are associated with immunodeficiency necrosis and/or crescent-shaped globules. The kit is useful for diagnosing systemic nephritis, and the kit contains two proteins of the present invention and a monoclonal protein of the present invention. Antibodies, miniroba oxidase, and monochromes against miniroba oxidase Contains local antibodies.

In another aspect, the invention provides a diagnostic kit for the detection of antibodies in biological fluid samples. The antibody is useful for diagnosing glomerulonephritis, and the kit contains two proteins of the present invention. White matter, miniloba-oxidase, complement C3, streptococcal antigen, type ■v collar Contains the NCI domain of the α3 chain of Gen., and DNA.

The term “immunodeficiency necrosis and/or crescentic glomerulonephritis” refers to the - Granulomas, microscopic polyarteritis nodosa, and early necrosis and/or crescentic This includes glomerulonephritis.

The compounds and methods of the present invention can be used to treat immunodeficiency NCGN (these symptoms are similar to sea urchin granuloma, microscopic polyarteritis nodosa, and early NCGN) and specific associated with other forms of glomerulonephritis that are present, easily interpreted, and easily quantified. Provides a means for detecting autoantibodies characteristic of several associated symptoms. anti-neutrophil cytoplasm Common detection methods for autoantibodies use autoantibody staining and indirect immunofluorescence. indirect exemption Judging epifluorescence findings requires considerable experience, and results vary from laboratory to laboratory. It might happen. Interpretation of results in assays of the invention can be performed using conventional solid phase or It relies on methods such as liquid-phase immunoassays. The results of these techniques are Very subtle differences in the patterns of antibodies bound to defined cellular structures can be identified and differentiated. In cases where it is necessary to It is much simpler to interpret than the given value. Additionally, it differs from indirect immunofluorescence assays. and the assay of the present invention can be quantitated.

The method of the present invention detects thyroid disease using antibodies that bind to autoantibodies characteristic of the disease. Either manufactured antigens or monoclonal antibodies directed against these antigens are used.

Where detection is based on the use of purified p29 or purified myelobe oxidase In the case of a method, the differences observed when using assays that use less purified antigen preparations. Different batches of antigen may provide different targets for autoantibody binding, such as There is no risk of For methods using monoclonal antibodies, the results are similarly specific. It's different.

Other features and advantages of the invention can be found in the following description of the preferred embodiments and in the claims. It becomes clear.

Description of preferred embodiments After first describing a brief description of the drawings, preferred embodiments of the invention will be described in detail.

drawing Figure 1 depicts the N-terminal sequences of p29 protein and several serine proteases. .

Figures 2 to 5 represent the results of direct solid-phase immunoassays of immunodeficient NCG\.

The following outlines two methods for detecting autoantibodies present in Wenner's granulomas. It is. The first method uses the antigen p29, which is recognized by autoantibodies, and the second The method uses p29 and monoclonal antibodies against p29, anti-p2 The production of 9 monochrome photoantibodies is described. Urchinina - Granuloma, microscopic nodular multiplication Autogenic arteritis and symptoms associated with immunodeficiency NCGN, including early NCGN. Two methods for detecting antibody characteristics are also provided. The first method is to use autoantibodies. Using two antigens, p29 and myeloperoxidase, which are recognized by The method uses these two antigens as well as monoclonal antibodies specific to each of them. Use your body. Method for preparing anti-myeloperoxidase monoclonal antibodies Describe.

Preparation of monoclonal antibody against 29kD protein Monoclonal antibody against 29kD Null antibodies were found in the lower limbs of female Ba1b/'c mice with a complete Freudian axis. was generated by immunizing with 1-0 μg of neutrophilic acid extract in Nyubando.

Neutrophilic acid extract was obtained by the method of Lockwood et al. ncet],: 716.1987). Simply put, orchid , lXlO' I5, then 0.2M sodium acetate buffer (pH 4). , 2) for 5 minutes at 0°C. (cells were diisopropylfluorofluorinated) If labeling with phosphate, add at 5mM and leave cells on ice for 10 minutes before washing. do. )　20. After centrifugation at 4°C for 20 minutes at 000g, the supernatant was adjusted to pH 7.4 or dialyzed against phosphate buffered saline, pH 7.4 (PBS).

The protein concentration in the sample was determined by Lowry et al. (J, Biol, Chem, 192:265.1951).

After three boosts over two weeks, axillary lymph nodes of injected mice were isolated. did. Lymph nodes are defined by Koh et al. (Nature, 256). :495-497.1975). It was fused with Maseru Line (American Thai Culture Collection). HAT For medium supernatants from hybridomas after 10-14 days of growth in selective medium, Anti-neutrophil activity was tested by Western blot analysis. Sea urchin sea urchin - granuloma self Western blot analysis of autoantibodies was performed as follows. as above The prepared acid extract (25 u g/lane) was added to Laemmli (Laemml 1) (N Sodium dodecyl sulfate described by Ature 227:680.1970) The cells were separated by polyacrylamide gel (SDS-PAGE) electrophoresis. to Towbin et al. (Proc, Nat 1., Hecad, Sc I, USA 76:4350.1979) protein from unstained Kel. White matter was electrophoretically transferred to nitrocellulose filters. Cut the membrane into strings , and then stained with serum from the patient (1.10 dilution), and further stained with bitinylated secondary antibody. color and form an avinone-biotin-peroxidase complex. combine The antibody is based on chromogen>3-amino-9ethylcarbamate, a peroxodase substrate. Detected by Sol.

Bands identified by sea urchin-granuloma autoantibodies (from serum) migrate together A monoclonal antibody that stains the 29kD band (p29) was selected. desired A hybridoma with an activity of Local antibody (mAb), IF5, was successfully isolated.

Monoclonal anti-p29 antibody, IF5 characteristics m, Ab, IF5, Western In the plot, it reacted with the 29kD nokundo derived from neutrophils, and the Indirect immunofluorescence staining identical to autoantibodies <1° that produced a turn Indirect immunofluorescence analysis was performed as follows. Anti-neutrophil cytoplasmic antibodies Indirect immunofluorescence using normal human neutrophils by cell centrifugation and ethanol fixation Detected by. Neutrophils are produced using Ficolu/S Ibakku gradient (Fic o1 Hypaque gradients) (Pharmacia, Biscatau) Boyum (Scand, J.) Clin, Lab, Invest, 97:77.1968) It was withdrawn by hypotonic lysis. Cell centrifugation preparations were prepared from Noyandong Southern (Sha) ndon 5outhern) cell centrifuge (Chesh + (Re) Ltd., UK). Each preparation was prepared for 5 min in 100% ethanol. Fix for minutes, dry, and incubate for 1 hour at room temperature (RT) with serum (116 dilution). Nki.

I bet. After washing twice, the cells were incubated with fluorescent sheep anti-human Ig (Memroy). .

y), Souvli>gtweild, VA, for 60 minutes at room temperature and washed. , and then observed using a fluorescence microscope.

Serum was obtained from 10 patients diagnosed with sea urchin granuloma. clinical Generally speaking, all cough patients, with or without rapidly progressing renal cough dysfunction, tract or upper respiratory tract disease (nasal erosions, sinusitis, hemoptysis). Pathologically, 3 A human patient had features of a lesion of necrotizing granulomatosis on nasal biopsy.

The remaining 7 patients had insufficient or no immunoglobulinosis or no immunoglobulin deposits. nasal vasculitis or pulmonary hairs with or without necrotizing or crescentic glomerulonephritis He had pathological evidence of tubulitis. Serum was also obtained from normal volunteers. all Serum was stored at -208C until use.

Normal neutrophil lysis was performed as described above for serum from the 10th batch of sea urchin granuloma. The lysates were screened for the presence of reactive autoantibodies. The serum of all patients is 1 Obtained from tissue specimens within 1 month. Serum from all 10 patients contained the 29kD antigen (p2 9) and cytoplasmic staining in ethanol-fixed neutrophils. A pattern arose. All sera from 200 normal people had anti-p29 antibodies. I didn't.

Purification of 29kD antigen (p29) By using the mAb IF5, Schneider et al. using the method of (J, Biol, Chem, 257:10766, Ko982). p29 was affinity purified. ], E8 (of the IgG1 subclass) is a dimethyl Binds to cephalometric suprotivin A beads coupled with rubimelimidate did. 10 m of immobilized monoclonal antibody-induced sepharose beads 1 column was roughly washed, then 30 mg of neutrophilic acid extract (details are given above) (prepared as described above) for 3 hours at room temperature. The column is Wash with 5x bed volumes of PBS, then 500mM sodium chloride. The cells were washed with 5 times the bed volume containing PBS. After re-equilibration with PBS, column with 0.2M citric acid, pH 2.75], eluted in both ml and concentrated with Tris salt. Immediately neutralized using a group of 7. The eluted protein was analyzed by spectrophotometry at OD 280. Detected by a meter. Pool the desired fractions and protein A-Sepharo (to remove contaminating amounts of contaminating mAb).

Concentrate the pooled fractions, and use Corono-Oshiba soge against distilled water. Dialyzed.

700 gg of protein was recovered in the eluate.

Antigen recognized by 1E8 and affinity purified under non-reducing conditions Migrated as three closely spaced bands on 5DS-PAGE with a major component of 29kD. did. The purified antigen was isolated from patient serum in Western blotting. It reacted with autoantibodies, but this indicates that the autoantibodies of sea urchin granuloma and IF5 It was confirmed that what was recognized was the same. In isoelectric focusing gel, p29 had an isoelectric point of 92 to 9.4.

p29 was shown to be a novel serine brotinase as described below.

Applied Biosystems model 10 μg of purified p29 for 20 cycles using Le 470A was subjected to Edman decomposition. Since one N-terminal sequence was obtained (Figure 1), 5D Molecular heterogeneity of purified proteins on S-PAGE is due to the isoforms of one protein. It was suggested that this may reflect the national biomedical Search Foundation (National Biomedical Re5 Foundation) and Swiss Protein Databanks ( Homology search using Swiss protein data, banks) A novel protein whose deduced sequence has significant homology with serine brotinases. It became clear that the quality was In particular, all serine brotinase catalytic Two hydrophobic residues (isoleucine and and valine) are present at the N-terminus. In addition, non-variable residues glycine (4th) and Proline (13th position) is present on p29. p29 exists in two early granules. What is neutrophil serine proteinase, leukocyte elastase, and categorical protein G? It was very different. Like leukocyte elastase and cathepsin G, p29 is It also bound to the radiolabeled DFP described above.

Determination of the N-terminal sequence was performed as follows. Steam 100 μg of purified p29. Dialyze endlessly against distilled water, shrink to 200 μm, and convert 20 μm into 5DS-PAG. E, dry plotted on Imopilone-P (Millivore) and indo ink. It was subjected to staining. Cut out the main p29 band with a safety razor blade and using an Abrid BioStems model 470A sequencer. It was subjected to 0 cycles of Edman degradation. PTH derivative is Cyno Kara (18M) and Permaphase ETH precolor Gradient elution (solvent A, 0 mM sodium acetate, pH 5, 5, 5% V/V tetrahydrofuran, solvent B acetonitrile: gradient: ], 1-48% Analysis was performed by HPLC for 20 minutes at a flow rate of 1 ml/min). Is it a cut out band? The N-terminal sequence obtained from was the same as

3H-DFP binding assay was performed as follows. Monoclonal antibody I By using E8 in a Sandwich radioimmunoassay, 3H - Binding of DFP to p29 was detected. IE8 Sodium acetate cut for ascites (cut) to 10ug/ml in PBS and 35μl/well for 1 hour. Incubate in 96-well polyvinyl microtiter plates at 7°C. I started. Unbound binding sites were blocked with 1% non-fat dry milk. Above An acid extract of neutrophils prepared without sea urchin DFP was diluted to 100 μg/ml. , and using 'H-DFP (3uCi/μM to 3.3nM, NEN), Incubated for 30 minutes at room temperature. Next, the extract (35 μl/well) was Wells pre-coated with F5 or irrelevant mAb or anti-MPOmA was added as a control to wells pre-coated with b. Incubate for 4 hours at room temperature. After baking, the wells were washed with PBS, dried, excised and exposed to beta fluorescence. Soaked and counted in a beta counter. Tritiated DFP is mAb I Bound only to wells containing F5.

Isoelectric focusing was performed in a horizontal gel apparatus (Hoef 7-( ) (06ffer) Scientific) to adjust the pH range to 3,5- It was carried out at 11. Gels were run at 2.5 mA constant current for 16 hours at 4°C. Fixed, stained with Coomassie Blue R-250, and destained.

Proteins were obtained from acid extracts and after washing the obtained proteins were exposed to test serum. stop The retained autoantibodies are detected using anti-human autoantibodies conjugated to markers.

Microtiter wells were pre-coated with mAb IF5 ammonium sulfate cut. exposed to neutrophil-acid extract (extracted as above) and incubated for 4 hours. Incubate. Wells were washed with PBS and 110 μm in a total volume of 35 μm. Exposure to test serum diluted to 0. After 60 minutes at room temperature, the wells were washed with PBS and and 1251-labeled sheep anti-human rg antibody (pre-neutralized to mouse IgG). ) to develop color. Cut out the wells, dry them, and count them in a gamma counter. Ru. ``5I-labeled human anti-human Ig antibody is an enzyme-conjugated anti-human autoantibody. Alternatively, other radio-like or non-radio-like markers may be substituted.

This assay consists of mAb IF5 (or equivalent) bound to microtiter wells. positive control samples (e.g., from a patient diagnosed with sea urchin granuloma) Serum) and other reagents needed to perform the assay. In this assay, purified p29 is used to obtain autoantibodies from the test serum. do. The autoantibodies obtained are made using anti-human autoantibodies conjugated with markers. detected.

p29 is affinity purified from neutrophil-acid extracts as described above. refined p29 is coated onto microtiter wells. Serum from patient (1:1 00 dilution) to the microtiter wells and incubated for 60 min. Cubated. Microtiter wells were washed with PBS and radiolabeled, Color was developed with anti-autoantibodies conjugated to enzymes or other labels.

This assay consists of p29 (or other suitable protein) bound to microtiter wells. matrix), positive control samples (e.g., serum from a patient diagnosed with sea urchin granuloma), ) and other reagents necessary to perform the assay. Ru.

The discovery of the production of additional monoclonal antibodies against the p29 protein - Circulating autoantibodies in the serum of patients with granulomas are directed against the protein we identified, p29. and enable the routine production of p29-specific monoclonal antibodies. That is. Such antibodies may be prepared by the methods described above or by comparable analogous methods below. can be produced.

Dissolves neutrophils in serum from any patient with Urchinona granuloma, and results in The resulting immunoprecipitate is brought into contact with the immunoprecipitate, and this precipitate collects the complex of p29 protein and antibody. include. By using this immunoprecipitate, animals such as mice can be immunized. , many of these animals produce antibodies against the p29 protein. Then, like that Normal and favorable Screened for monoclonal antibodies that bind to lysates from neutrophils. Ta. Recognition of PBS protein was confirmed by Western blotting as described above. be done.

Cloning of PBS gene The above information on the N-terminal amino acid sequence of p29 is based on the gene encoding the protein. makes cloning routine, especially in view of the small size of the protein.

Standard techniques, i.e., screening cDNA libraries from neutrophils, By using the terminal sequence information, synthetic oligonucleotides are created and By using oligonucleotides, the cDNA encoding p29 protein is can get.

Autoantibodies in both cases (onset arteritis and early NCGN) are characterized by anti-p29 autoantibodies. Assay for autoantibodies and assay for anti-miniloba-oxidase autoantibodies It can be detected by combining the

- Cal Bjochem Behring, San Diego, CA) of the anti-p29 monoclonal antibody outlined above by using Anti-MPO antibodies can be prepared for. Purified anti-MPO monoclonal antibody Diagnosed with necrotizing and/or crescentic glomerulonephritis using Patient serum can be screened. Selected anti-MPO monoclonal antibodies Most of the serum samples reacted with indirect immunofluorescence of ethanol-fixed normal neutrophils. The assay should yield a nuclear or perinuclear staining pattern (fan Desired Hybridomas with reactivity can be subcloned; Monoclonal antibodies use Protein-A Sepha 0-chromatography ms by standard techniques. Alternatively, commercially available (Dako) Co., Ltd., Zanta Barbara, CA) Tuclonal anti-myeloperoxidase antibody can be used. .

Indirect homologous immunotherapy for immunodeficiency NCGN autoantibodies! Say this anose 1' Neutrophil acid was detected using monoclonal anti-p29 and anti-MPOυ. Manually extract proteins from the extract After washing, expose the obtained white matter to test serum 7 , the retained autoantibodies were detected using an anti-human Ig antibody conjugated with a marker. Ru.

Microtiter wells were filled with mAb IF5 or purified anti-! vIPo　m Pre-coat ammonium sulfate cut of Ab and pre-coat with neutrophilic acid extract (as above) The ink was then exposed to the first ink prepared for 4 hours. PB well Wash with S and expose to test sample diluted 1100 in a total volume of 35 μl.

After 60 minutes at room temperature, the wells were washed with I) B S and +251. -labeled human Color is developed with an anti-Hi1-1g antibody (pre-neutralized to IgG). cut the well Remove, dry, sieve and count using a gamma counter. +25I-labeled human anti- Human Ig antibodies can be enzyme-conjugated anti-HiIg antibodies or other radio-like markers. Cars or non-radial-like markers may be substituted.

This assay consists of mAb IF5 (or or similar substrate), anti-MPO mAb bound to other microtiter wells ( or similar substrates), positive control samples (e.g. from patients diagnosed with immunodeficiency), Serum) and other reagents needed to perform the assay. 3. Alternatively, both mAbs can be placed in one well. Such a kit detects the presence of autoantibodies associated with immunodeficiency NCGN. make it possible.

Immunodeficiency NCGN autoantibodies [n-attached solid-phase immunoassay] In this case, autoantibodies were introduced from the test serum using purified 1)29 and purified VMPO. hand. The human-made autoantibodies are marker-conjugated anti-human 1-1g antibodies. detected using

p29 was affinity purified from neutrophil-acid extracts as described above and - Co) into microtiter wells. The second microtiter well is Coated with purified MPO. Serum from the patient (35 ul of 1:100 dilution) was microtiter wells and incubated for 60 minutes. Ma The microtiter wells were washed with PBS [7, and the radiolabel, enzyme binding, Or, the color was developed with anti-1g antibody with other labels.

This means that p29 (or other suitable substrate), positive control samples (e.g., diagnosed with immunodeficiency NCGN. A complete package containing the reagents (reagents) and other reagents needed to perform the assay. Served. Alternatively, both mAbs can be placed in one well.

This key contains substances for the detection of anti-globular basement membrane (anti-GBM) antibodies. Good too. For example, glomerular basement membrane collagen degradation products can be collected in microtiter wells. (Wilson et al., Kidn+δy I nt　6・114. A., 1974).

Example of direct solid-phase immu-71 assay of immuno7z foot NCGN autoantibody 7' Four groups of individual sera were tested using a solid phase immunosorbent. glue Group A consisted of 42 patients with immunocompromised NCGN, Group B consisted of 200 blood bank donors. Blood relatives, group C positive for anti-GBM antibodies] 8 donors, group D positive for anti-GBM antibodies Diagnosed with M, but proven to have immunodeficiency NCGN or anti-08M nephritis. There were 62 patients who did not. 1 p29 and MPO, J Oim 2 Noah Assose 1) 29 are listed above. es) et al., Blood 74:], 88.1989). Purified from crude acid extract of granulocytes isolated by affinity chromatography. It was. Purified fMPO was purchased from Calbiochem (San Diego, CA). . p29 and MPO were each dissolved in milliliters of poric acid buffer salt pH 8.1 (BBS). Diluted to 10 macrograms and 507 micrograms per needle.

Add 35 μl/well of p29 and MPO to a polyvinyl microtiter plate. Costar 5 scientific , Cambrino/, MA) for 1 hour at 37°C. non-singular To assess biological binding, some wells were incubated with BBS alone as a control. 1. Unbound sites were added to wells with 1% non-fat dry milk in BBS for 1 hour 3. Blocked by addition at 7°C. Next, dilute the wells with various dilutions. Ink with 435 µl of blood from 100% of the ink in triplicate for 2 hours (or overnight) at room temperature. It was the first time. After washing with BBS, the wells were injected with 35 ml of 1251-labeled Hira/anti- -Human immunoglobulin horn (1.Tug of 1500CPM/ng, with -'mri) The ink was applied to the paper. After the final wash, 4+, dry the wells and activate comma. were counted. Two standard positive control m111 samples, one with anti-])29 activity and the other had anti-MPO activity in 8 series from 116 to 1:2048. Serial dilutions were made in each assay. Each test serum was diluted 1-16. was assayed. Take the average of the three counts and obtain only the control wells. The average count number of each sample obtained in p 29 and p, j)) ○ deducted from the actual amount.

A standard curve was constructed from 8 values of the positive control. Two standard positive undiluted serum samples has an indeterminate activity of 128 units, and each serial 1-:2 dilution has many units of activity. It had 1°5 times. Next, count each test serum (subtract non-specific counts). The unit of anti-p29 or anti-MPO activity was determined by reading the standard curve. The number of knits was determined. The characteristic curve operated by Renover is the immunodeficiency NCGN. and mean and standard log of anti-p29 and anti-MPO antibody titers of normal controls. derived from the standard deviation (Sox et al., Medical Decisi on Making, Butterworth, 1988) o anti-p29 and while retaining the 99% combined specificity of the anti-MPO antibody assay. The Cutoff value gives the most likely combined sensitivity Selected anti-p29 and anti-MPO antibodies, as described above, contain less than 1 unit. Calculated on an activity scale ranging from 128 units or more. Group A (immune Antibody titers for group B (deficit NCGN) and group B (blood bank donors) are shown in the figure, respectively. 2 and plotted in Figure 3. As described in the methods, these two groups loop and used in 99% of anti-p29 and anti-MPO antibody assays. While retaining the combined specificity, we identified 95% of immunodeficient NCGN and -1) Combining 15 units of 29 and 25 units of anti-MPO All patients except 2 in group 3 and group 48 (immunocompromised NCGN) who were assessed for susceptibility ＼ indicates positive for either anti-p29 antibody or anti-MPO antibody 1. Ta. 2) One of the negative individuals tested positive for p in the withdrawal serum collected after 3 months. It was found that he had antibodies against 29. Urchinona - the only one diagnosed with granuloma The rfn serum of the patient was not positive for anti-p29 antibodies. This patient's serum is anti-M The patient was positive for PO antibodies. All samples from blood bank donors are tested for both antibodies. The results were negative.

Having decided to use the cutoff value determined above, the two other groups The test results of samples C and D were analyzed. Group C has anti-GBM antibodies in the study. It consisted of 18 patients who were positive for. Immunofluorescence results of patients with stiffness showed anti-G Special for BM neutrophils? :1. It was -7 at Guj. Group D was diagnosed with RP C:: However, no correction was found for immunodeficient NCGN or anti-GBM neutrophils. It consisted of 62 patients.

Of the 18 people in group C, 2 had anti-p29 antibodies and 6 had anti-MP was found to have O antibodies (Figure 4). In Group D, 62 people Two of them were both calculated to be positive for anti-p29 antibodies (Figure 5). By using After reviewing the available clinical and experimental data, these two patients were diagnosed with NCG. It was concluded that N.

Overall, among those with clinical RPGN syndrome, anti-p29 or anti-MPO By combining anti-GBM antibody positive with anti-GBM antibody negative, renal No organ specimen is available (although it does not provide a highly reliable indicator for the diagnosis of immunodeficiency NCGN). provided.

how to use By using antibodies against D29 protein and/or p29, sea urchin The presence of autoantibodies can be tested to aid in the diagnosis of granulomas. myeloper Combination of antibodies against oxidase and/or myeloperoxidase By using p29 and/or antibodies against p29, The presence of autoantibodies can be tested to aid in the diagnosis of NCGN deficiency. p29 By using the protein with other antigens, the presence of antibodies associated with glomerulonephritis can be tested.

Other aspects Pond embodiments are within the scope of the following claims. For example, p29 or an anti-p29 Use the body as part of a kit using direct or indirect immunoassays detects antibodies associated with the full blood spectrum of symptoms associated with glomerulonephritis. Can be detected. This antigen includes: myeloperoxidase, complement C 3 (Robbins) Pathological Ba5is.

f　Diseases, 5 anders, p. 1029), streptococcal antigen Causer, American J, Kidney Diseases es 11:449.1988), type IV collagen α3 chain NCI Main (Ba Islang-CWe: S la n, :ICr>et al., p. 7 0 C, p: at:, Acad, Sci,, USA 81: 1544.19 84), DN, A (Condemi et al., JAMA 259:2 920), and guidance for the detection of said autoantibodies.

2.5 units anti-MPO Anti-MPO antibody titer (units) 2.5 units anti-MPO Anti-MPO antibody titer (units) FIG.4 2.5, 2nd anti-MPO Anti-MPO antibody titer (units) FIG.5 2.5 units anti-MPO Anti-MPO antibody titer (Conit) procedural arrest Date: September 1992

Claims (10)

[Claims] 1. (a) can be isolated from neutrophils; (b) by SDS-PAGE about 2 determined to have a molecular weight of 9 kD; (c) bound to diisopropylfluorophosphate; (d) have an isoelectric point of about 9.2 to about 9.4; (e) wedge able to bind to autoantibodies present in the serum of people suffering from granulomas. ,and (f) Characteristics of the N-terminal aminoacyl sequence [there is a sequence] (SEQ ID NO: 1) A virtually pure protein with 2. A monoclonal that can form an immune complex with the protein according to claim 1. antibody. 3. How to detect autoantibodies in biological fluid samples to aid in the diagnosis of Wesiner's granuloma The law is: (a) contacting said biological fluid sample with the protein of claim 1; and (b) detecting the immune complex formed in step (a), determining the shape of the immune complex; said method, wherein said composition is indicative of the presence of autoantibodies. 4. Wesiner - How to detect autoantibodies in biological fluid samples to aid in the diagnosis of granulomas The law is: (a) Reactive with the monoclonal antibody according to claim 2 and the monoclonal antibody (b) providing an immune complex with an antigen; (b) introducing said immune complex into a biological fluid sample; and (c) detecting the binding of the autoantibody to the immune complex. and the formation of said immune complex is indicative of the presence of autoantibodies in the biological fluid sample. The above method. 5. A vector containing a DNA sequence encoding p29 protein. 6. Biological fluid samples useful in the diagnosis of immunodeficiency necrosis and semiarcuate glomerulonephritis A method for detecting autoantibodies in: (a) contacting the sample with the protein of claim 1; (b) contacting the sample with the protein of claim 1; - contacted with an oxidase; and (c) formed in step (a) or (b). comprising detecting immune complexes, the formation of which is detected in a biological fluid sample. The method is an indicator of the presence of autoantibodies. 7. Biological A method for detecting autoantibodies in a liquid sample, comprising: (a) the monoclonal antibody according to claim 2; and an antigen reactive with said monoclonal antibody: (b) Myeloperoxidase and monochrome for myeloperoxidase Provide immune complexes with null antibodies: (c) contacting the following sample with the immune complex of step (a): (d) the immunization of step (b); contacting the complex with the sample: and (e) the immunoconjugate of step (a) or (b). It consists of detecting the binding of autoantibodies to the body, and the formation of said immune complexes is Said method is indicative of the presence of autoantibodies in a biological fluid sample. 8. A biological protein comprising the protein according to claim 1 and myeloperoxidase. Diagnosis of immunodeficiency necrosis and/or crescentic glomerulonephritis present in fluid samples Diagnostic kit for detecting autoantibodies useful for. 9. The protein according to claim 1, the monoclonal antibody according to claim 2, myelopathy - Monoclonal antibodies against oxidase and myeloperoxidase immunodeficient necrosis and/or crescentic threads present in biological fluid samples A diagnostic kit for detecting autoantibodies useful in the diagnosis of bulbar nephritis. 10. The protein according to claim 1, myeloperoxidase, complement C3, chain globules Contains bacterial antigen, NC1 domain of α3 chain of type IV collagen, and DNA , to detect antibodies present in biological fluid samples that are useful in diagnosing glomerulonephritis. diagnostic kit.